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      Extracellular Vesicle Quantification and Characterization: Common Methods and Emerging Approaches

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          Abstract

          Extracellular vesicles (EVs) are a family of small membrane vesicles that carry information about cells by which they are secreted. Growing interest in the role of EVs in intercellular communication, but also in using their diagnostic, prognostic and therapeutic potential in (bio) medical applications, demands for accurate assessment of their biochemical and physical properties. In this review, we provide an overview of available technologies for EV analysis by describing their working principles, assessing their utility in EV research and summarising their potential and limitations. To emphasise the innovations in EV analysis, we also highlight the unique possibilities of emerging technologies with high potential for further development.

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          Most cited references100

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          Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

          Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. Copyright © 2012 Elsevier Inc. All rights reserved.
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            Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis.

            Exosomes are naturally occurring biological nanovesicles utilized by tumors to communicate signals to local and remote cells and tissues. Melanoma exosomes can incite a proangiogenic signaling program capable of remodeling tissue matrices. In this study, we show exosome-mediated conditioning of lymph nodes and define microanatomic responses that license metastasis of melanoma cells. Homing of melanoma exosomes to sentinel lymph nodes imposes synchronized molecular signals that effect melanoma cell recruitment, extracellular matrix deposition, and vascular proliferation in the lymph nodes. Our findings highlight the pathophysiologic role and mechanisms of an exosome-mediated process of microanatomic niche preparation that facilitates lymphatic metastasis by cancer cells.
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              High Levels of Exosomes Expressing CD63 and Caveolin-1 in Plasma of Melanoma Patients

              Background Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up. Methodology/Principal Findings We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504±315) or caveolin-1 (619±310) were significantly increased in melanoma patients as compared to healthy donors (223±125 and 228±102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group. Conclusions/Significance We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.
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                Author and article information

                Journal
                Bioengineering (Basel)
                Bioengineering (Basel)
                bioengineering
                Bioengineering
                MDPI
                2306-5354
                16 January 2019
                March 2019
                : 6
                : 1
                : 7
                Affiliations
                [1 ]Department of Pathology, Erasmus Optical Imaging Centre, Erasmus MC, 3015 GE Rotterdam, The Netherlands; t.hartjes@ 123456erasmusmc.nl
                [2 ]Department of Chemical Engineering, Delft University of Technology, 3015 CD Delft, The Netherlands; S.Mytnyk@ 123456tudelft.nl (S.M.); V.vanSteijn@ 123456tudelft.nl (V.v.S.)
                [3 ]Department of Urology, Erasmus MC, 3015 CD Rotterdam, The Netherlands; g.jenster@ 123456erasmusmc.nl
                Author notes
                [* ]Correspondence: m.vanroyen@ 123456erasmusmc.nl
                [†]

                Authors contributed equally.

                [‡]

                Authors contributed equally.

                Author information
                https://orcid.org/0000-0002-0110-9433
                https://orcid.org/0000-0002-8658-2552
                https://orcid.org/0000-0002-6814-0996
                Article
                bioengineering-06-00007
                10.3390/bioengineering6010007
                6466085
                30654439
                5ff8fb52-d741-416d-bb4f-a0d5e5ca6523
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 23 December 2018
                : 15 January 2019
                Categories
                Review

                extracellular vesicles (evs), methods for ev analysis,emerging technologies

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